Methods and compositions for selective modulation of vascularization

ABSTRACT

The invention provides methods and compositions for modulating vascularization and angiogenesis, including selective modulation of stages of multi-step vascularization and angiogenesis.

RELATED APPLICATIONS

This application is a non-provisional application filed under 37 CFR1.53(b)(1), claiming priority under 35 USC 119(e) to provisionalapplication No. 60/463,226 filed Apr. 16, 2003, the contents of whichare incorporated herein in its entirety by reference.

TECHNICAL FIELD

The present invention relates generally to the fields of molecularbiology and vascularization. More specifically, the invention concernsmodulators of angiogenesis, and uses thereof.

BACKGROUND

Stanniocalcin 1 (STC-1) is a secreted glycoprotein originally describedas a hormone involved in the calcium and phosphate homeostasis in bonyfishes. The mammalian homolog of this molecule has been identified asbeing highly upregulated in an in vitro model of angiogenesis as well ashighly expressed at sites of pathological angiogenesis (e.g. tumorvasculature). See, for e.g., Kahn et al., Am. J. Pathol. (2000),156(6):1887-1900. Its potential role in pathological conditions such ascancer has also been suggested. See, for e.g., Fujiwara et al., Int. J.Oncol. (2000), 16:799-804. Preparation of an antibody for a human STC-1peptide is reported in Koide et al., Biol. Pharm. Bull. (1998),21(12):1352-1355. However, the precise role of STC-1 in the angiogenesisprocess is unclear.

Numerous factors that promote or inhibit angiogenesis have beenidentified, reflecting the importance of the role of angiogenesis inhomeostasis and pathological conditions. See, for e.g., Folkman,Seminars in Oncol. (2002), 29(6) (Suppl. 16):15-18; Ferrara, Seminars inOncol. (2002), 29(6) (Suppl. 16):10-14. Well-known angiogenic promotersinclude vascular endothelial growth factor (VEGF) and hepatocyte growthfactor (HGF).

HGF is a mesenchyme-derived pleiotrophic factor with mitogenic,motogenic and morphogenic activities on a number of different celltypes. HGF effects are mediated through a specific tyrosine kinase,c-met, and aberrant HGF and c-met expression are frequently observed ina variety of tumors. See, for e.g., Maulik et al., Cytokine & GrowthFactor Reviews (2002), 13:41-59. Recent studies have shown HGF to be apotent growth factor implicated in wound healing, tissue regenerationand angiogenesis.

It was also recently reported that HGF, and more potently, HGF incombination with VEGF, synergistically induced vascular morphogenesis invitro and angiogenesis in vivo (1). The gene expression profile ofendothelial cells undergoing HGF and VEGF stimulated morphogenesis usingAffinetriX™ oligonucleotide arrays was also analyzed. The homodimericsecreted glycoprotein, Stanniocalcin-1 (STC-1), was shown as one of themost highly upregulated genes in this in vitro model (2). Intenseexpression of STC-1 was observed in the vasculature of colon carcinomas(2). However, the precise nature of the role(s) of STC-1 invascularization generally, and angiogenesis specifically, remainsheretofore unclear.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

DISCLOSURE OF THE INVENTION

Methods of the invention are based generally on the finding that certainangiogenic modulators selectively affect some stages of the angiogenicprocess but not others. It is also herein disclosed that certainangiogenic modulators are capable of affecting angiogenesis induced bysome angiogenic promoters but not others. Thus, these factors provideunique and advantageous targets for greater fine-tuning in designingprophylactic and/or therapeutic approaches against pathologicalconditions associated with abnormal angiogenic states. Accordingly, theinvention provides methods, compositions, kits and articles ofmanufacture for modulation of angiogenesis. The invention isparticularly useful for modulation of specific stages in the multi-stageprocess of angiogenesis. In particular, modulators and methods of theinvention are based on regulating certain stages of the angiogenesisprocess in disease states wherein abnormal angiogenesis occurs. Theinvention is also especially useful for modulation of extent or amountof angiogenesis, for example by inhibiting the angiogenic process, orsubset stages thereof, promoted by one angiogenic promoter but not theangiogenesis process promoted by another angiogenic promoter. Sinceangiogenesis is important in homeostasis and normal physiologicalfunctions, the ability to reduce but not eliminate (or substantiallyinhibit) angiogenesis would provide a great advantage in therapeuticdesigns wherein it may be desirable to reduce but not eliminateangiogenesis in a single tissue and/or subject. Hence, compositions andmethods of the invention provide unique advantages in overcomingdifficulties inherent in the complexities of the angiogenic process andthe related difficulties of therapeutically addressing disease statesassociated with pathological angiogenic states.

In one aspect, the invention provides methods of modulating angiogenesiscomprising administering to a cell undergoing the angiogenic process aneffective amount of stanniocalcin-1 (STC-1) or variant (as furtherdefined below) thereof. The cell undergoing the angiogenic process maybe present in an in vitro culture, an ex vivo culture or in vivo. In oneembodiment, the cell is an endothelial cell.

In one embodiment, migration or morphogenesis of the cell undergoing theangiogenic process (which is generally an endothelial cell) issubstantially inhibited. In one embodiment, the vascularization orangiogenic process of methods of the invention is associated withpresence of hepatocyte growth factor (HGF). Said HGF may be expressed inthe cell undergoing the angiogenic process, a neighboring cell and/orwhich is present systemically. Said HGF may also be providedexogenously. In some embodiments, expression or presence of hepatocytegrowth factor occurs prior to and/or parallels initiation of theangiogenic process.

In some embodiments, modulation by STC-1 or variant thereof in methodsof the invention is substantially inhibited by an antagonist of STC-1.In some embodiments, said antagonist is an antibody or fragment thereof.Such antagonists can be generated using methods known in the art,including those described herein. The invention also providesmonocloncal antibodies, including blocking antibodies useful in, forexample, methods of the invention. Antibodies of the invention includeantibodies produced by hybridoma clones designated 3E10.1F5.1D11 (ATCCDeposit No. PTA-5100), 21 F4.2F11.1H12 (ATCC Deposit No. PTA-5103),1F10.2F12.1E7 (ATCC Deposit No. PTA-5101), 18F1.1E6.1B 10 (ATCC DepositNo. PTA-5099) and 12C3.2F3.2D8 (ATCC Deposit No. PTA-5102). Theinvention also provides antibodies that bind to substantially the sameSTC-1 epitope as antibodies produced by these hybridomas. In someembodiments, antibodies of the invention bind to or at the same STC-1epitope as antibodies produced by these hybridomas. In some embodiments,antibodies of the invention compete with antibodies produced by thesehybridomas in binding to STC-1. In one embodiment, an antibody of theinvention is not an anti-peptide antibody. In one embodiment, anantibody of the invention does not bind to the sequenceCYNRLVRSLLEADEDTVSTI (SEQ ID NO:3). In one embodiment, an antibody ofthe invention does not compete for binding to STC-1 with an antibodythat binds to the sequence CYNRLVRSLLEADEDTVSTI (SEQ ID NO:3). Variabledomain sequences, including CDR sequences, of the antibodies describedherein can be easily determined using techniques known the art.Antibodies and immunoglobulin polypeptides comprising the heavy and/orlight chain variable domain sequences of the antibodies described hereinare also provided. In some embodiments, the invention providesantibodies and immunoglobulin polypeptides comprising the sequence of atleast one, at least two, or all three CDRs of the heavy and/or lightchain of the antibodies described herein. In one embodiment, an antibodyof the invention is a monoclonal antibody, which in some embodiments isa chimeric/humanized or human antibody, or functional fragment thereof.These antibodies and immunoglobulin polypeptides would be useful inmethods of the invention as long as they retain the STC-1 bindingcapability of the antibodies designated herein. Other antagonists areknown in the art, including antisense oligonucleotides, small moleculesand other inhibitory chemical compounds (such as organic and inorganicmolecules). These antagonists can be produced by methods known in theart, including those disclosed herein.

Many angiogenic factors have been identified, and often cells undergoingangiogenesis are exposed to more than one angiogenic factor. Yet, thedownstream effects of these factors in effecting angiogenesis are notfully elucidated. The present invention has identified a factor thatselectively modulates some but not all stages of angiogenesis, in theangiogenic response of cells to some but not all angiogenic factors.Accordingly, in another aspect, the invention provides methods ofselectively inhibiting angiogenesis promoted by a first angiogenicfactor but not angiogenesis promoted by a second angiogenic factor,comprising administering to a cell exposed to both factors an effectiveamount of STC-1 or a variant thereof, wherein said STC-1 or variantthereof inhibits at least one step of the angiogenic process promoted bythe first angiogenic factor but not the angiogenic process promoted bythe second angiogenic factor

In some embodiments of methods of the invention, the first and secondangiogenic factors both are capable of inducing endogenous expression ofSTC-1. In one embodiment, the first angiogenic factor is hepatocytegrowth factor. In another embodiment, the second angiogenic factor isbasic fibroblast growth factor (bFGF). In some embodiments, the firstangiogenic factor is hepatocyte growth factor and the second angiogenicfactor is vascular endothelial growth factor (VEGF) and/or bFGF.

In some embodiments of methods of the invention, STC-1 or variantthereof inhibits or substantially inhibits cell migration and/ormorphogenesis induced by or associated with exposure of the cell to thefirst angiogenic factor. In certain embodiments, STC-1 or variantthereof does not inhibit or substantially inhibit cell proliferation.

In some embodiments of methods of the invention, STC-1 or variantthereof does not inhibit or substantially inhibit cell migration and/ormorphogenesis induced by or associated with exposure of the cell to thesecond angiogenic factor.

Generally, and preferably, the cell undergoing angiogenesis in anymethod of the invention is of endothelial origin.

In another aspect, the invention provides polypeptides comprising anamino acid sequence of at least 80%, 85%, 90%, 95%, 98%, 99% identity tothe sequence of human STC-1 (as provided herein in FIG. 6), wherein aneffective amount said polypeptide substantially inhibits at least one,but not all, of the stages of multi-stage angiogenic process. Theangiogenic process is preferably induced by or associated with thepresence of hepatocyte growth factor. Expression of polypeptides of theinvention may be induced by any one or combination of factors, includingfactors in the group consisting of IL-1β, bFGF, HGF, IL-6 and IL-4.

In some embodiments, polypeptides of the invention inhibit orsubstantially inhibit endothelial cell migration and/or morphogenesisbut do not inhibit or substantially inhibit endothelial cellproliferation.

In some embodiments, a polypeptide of the invention does notsubstantially reduce c-met phosphorylation, for example in anendothelial cell.

In another aspect, the invention provides polynucleotides encoding anyof the polypeptides of the invention.

In another aspect, the invention provides antagonists, such asantibodies (or fragments thereof), against STC-1. Examples of theseantibodies include those described herein. In some embodiments, theseantibodies are capable of neutralizing or blocking STC-1 activity orfunction, examples of which include antibodies described herein. Otherantagonists of STC-1 are also provided, such as small molecules,peptides, etc. These antagonists can be readily screened for by methodsknown in the art, including the assays and/or methods of screening, andbiological readouts as described herein.

In another aspect, the invention provides methods of substantiallyinhibiting vascularization (for example, angiogenesis) in a mammal (forexample, human), comprising administering to the mammal an effectiveamount of STC-1 or variant thereof. Said vascularization may be inducedby or associated with presence of hepatocyte growth factor.

In another aspect, the invention provides methods of screening for asubstance that selectively inhibits HGF-induced vascularization (forexample, angiogenesis), said methods comprising comparing extent ofinhibition when an endothelial cell is exposed to said substance withthe extent of inhibition when the cell is exposed to STC-1 or an STC-1agonist. Thus, in one aspect, the invention provides STC-1 agonists,such as a small molecule (including peptides, antibodies or fragmentsthereof, organic and inorganic molecules) that mimics and/or enhancesthe function/activity of STC-1 in inhibiting angiogenesis as describedherein.

In another aspect, the invention provides methods of screening for asubstance that selectively promotes HGF-induced vascularization (forexample, angiogenesis), said methods comprising comparing the extent ofvascularization (for example, angiogenesis) when an endothelial cell isexposed to said substance with the extent of vascularization (forexample, angiogenesis) when the cell is exposed to an antagonist ofSTC-1 (for example, an antibody as described herein).

In another aspect, the invention provides methods of promotingvascularization (for example, angiogenesis) in a mammal (for example,human), comprising administering to the mammal an effective amount of anantagonist of STC-1 or variant thereof. Said vascularization may beinduced by or associated with presence of hepatocyte growth factor.

In another aspect, the invention provides methods of diagnosis forconditions associated with activation of the HGF/c-met pathway orHGF-induced angiogenesis, comprising detecting presence and/or amount ofSTC-1 or STC-1 activity in a sample of interest. As appropriate, areference sample (for e.g., that comprising a normal correspondingtissue) known or thought to exhibit a baseline amount of STC-1 or STC-1activity may be used in these methods. These methods are particularlyuseful where it is desirable to detect and/or quantitate downstreameffects/events of an axis known or suspected to be involved in apathological condition associated with HGF/c-met induced angiogenesis.For example, presence and/or degree of these downstream effects (such asSTC-1 activation/expression/activity) may serve as better or moreaccurate indicators of extent of the pathological condition. Thus, forexample, degree of STC-1 induction/expression/activity may provide anindication of the role/extent of angiogenesis in an HGF/c-met inducedpathological condition.

In methods of the invention, STC-1 may be provided as a polypeptide(including any described herein) or polynucleotide encoding saidpolypeptide. Examples of a polynucleotide encoding STC-1 include apolynucleotide comprising the nucleic acid sequence of SEQ ID NO:1 (FIG.6), a polynucleotide encoding the amino acid sequence of SEQ ID NO:2(FIG. 6) and a polynucleotide encoding an STC-1 variant (as describedmore extensively below).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. STC-1 mRNA and protein are selectively upregulated in 3-Dgel environments. A. STC-1 mRNA from HUVECs cultured in (3-D) or on thesurface of (monolayer) collagen gels in BM supplemented with HGF (200ng/mL) and VEGF (200 ng/mL). Duplicate samples were analyzed byquantitative RT-PCR (Taqman) as described in Example 1. Results areexpressed as the ratio of STC-1 mRNA level to the level of Cyclophillin,a housekeeping gene, in the same sample. B. STC-1 protein from HUVECscultured in (3-D) or on the surface of (monolayer) collagen gels in BMsupplemented with HGF (200 ng/mL) and VEGF (200 ng/mL). STC-1 proteinwas determined by ELISA as described in Example 1. Values shown are themean±S.E.M., n=8. * significantly different from value at t=0, p<0.05

FIG. 2. STC-1 inhibits endothelial cell migration A. The migratoryresponse of HUVEC to 20 ng/mL of HGF in the presence of native (solidbars) or boiled rSTC-1 (hatched bars) at the indicated concentrationswas determined. Data are expressed as the percent of the HGF controlcell migration index and are the mean±S.E.M., n=4. Data shown arerepresentative of three independent experiments. * Significantlydifferent from HGF alone, p<0.05. B. The inhibitory effects of STC-1 onHGF (20 ng/mL)-induced migration are blocked by the monoclonal antibody2734 (25 μg/mL) (produced by hybridoma clone 21F4.2F11.1H12, which hasATCC Deposit No. PTA-5103), but not by an isotype-matched nonimmune IgG.Data are expressed as cell migration index (number of cells/2× field)and are the mean±S.E.M., n=4. Data shown are representative of threeindependent experiments. * Significantly different from control. ++Significantly different from HGF alone, p<0.05.

FIGS. 3A-C. STC-1 reduces endothelial cord formation on Matrigel inducedby 20 ng/ml HGF. A. HGF+boiled STC-1 (250 ng/ml) B. HGF+native STC-1(250 ng/ml) C. Quantitation of cord formation in the presence of boiledSTC-1 (solid bars) or native STC-1 (hatched bars). Data shown are themean±SEM network area/well of three independent experiments. *significantly different from boiled control.

FIG. 4. STC-1 does not inhibit HGF induced c-met phosphorylation. HUVECwere pretreated for 30 min with 5 μg/ml STC- 1 or 5 μg/ml boiled STC-1,then challenged with HGF (100 ng/ml) for 15 min. Lysates were subjectedto immunuprecipitation for c-met, separated by gel electrophoresis, andtransferred to nitrocellulose. The resulting blots were immunoblottedwith antibodies to phosphotyrosine (pTyr)(top) or c-met (bottom).

FIGS. 5A-B. STC-1 is upregulated in the hind limb ischemia model ofangiogenesis. A.STC-1 and CD31 mRNA in gastrocnemius muscles removedfrom mice following sham surgery or femoral ligation. B. HGF mRNA ingastrocnemius muscles removed from mice following sham surgery orfemoral ligation. Values are expressed as the mean±S.E.M., n=6 mice pertime point and treatment.

FIGS. 6A-C. STC-1 sequences. Nucleic acid (SEQ ID NO:1) and amino acid(SEQ ID NO:2) sequences of human STC-1.

MODES FOR CARRYING OUT THE INVENTION

The invention provides methods, compositions, kits and articles ofmanufacture for selective modulation of vascularization/angiogenesis.

Details of these methods, compositions, kits and articles of manufactureare provided herein.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., ed., 1994); “A Practical Guide to MolecularCloning” (Perbal Bernard V., 1988).

Definitions

The term “STC-1 polypeptide”, as used herein, refers to a polypeptidecomprising the amion acid sequence listed in FIG. 6 (SEQ ID NO: 2) andSTC-1 polypeptide variants (as further defined below). An STC-1polypeptide may be isolated from a variety of sources, such as fromhuman tissue types or from another source, or prepared by recombinant orsynthetic methods.

“STC-1 polypeptide variants,” as used herein, refers to a polypeptidehaving at least one of the biological activities of the STC-1polypeptides as described herein (including at least an ability toinhibit at least one stage of the multi-stage HGF-induced angiogenicprocess) and having at least about 80% amino acid sequence identity withSEQ ID NO: 2 (as listed in FIG. 6). Ordinarily, an STC-1 polypeptidevariant will have at least about 80% amino acid sequence identity,alternatively at least about 81% amino acid sequence identity,alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to SEQ IDNO: 2 (FIG. 6). Ordinarily, STC-1 polypeptide variants are at leastabout 10 amino acids in length, alternatively at least about 20 aminoacids in length, alternatively at least about 30 amino acids in length,alternatively at least about 40 amino acids in length, alternatively atleast about 50 amino acids in length, alternatively at least about 60amino acids in length, alternatively at least about 70 amino acids inlength, alternatively at least about 80 amino acids in length,alternatively at least about 90 amino acids in length, alternatively atleast about 100 amino acids in length, alternatively at least about 150amino acids in length, alternatively at least about 200 amino acids inlength, alternatively at least about 300 amino acids in length, or more.

“Percent (%) amino acid sequence identity” with respect to the STC-1polypeptide sequence (i.e., SEQ ID NO: 2) identified herein is definedas the percentage of amino acid residues in a candidate sequence thatare identical with the amino acid residues in the specific STC-1polypeptide sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. Forpurposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2,wherein the complete source code for the ALIGN-2 program is provided inTable 2 below. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc. and the source code shown in Table 2 belowhas been filed with user documentation in the U.S. Copyright Office,Washington D.C., 20559, where it is registered under U.S. CopyrightRegistration No. TXU510087. The ALIGN-2 program is publicly availablethrough Genentech, Inc., South San Francisco, Calif. or may be compiledfrom the source code provided in Table 2 below. The ALIGN-2 programshould be compiled for use on a UNIX operating system, preferablydigital UNIX V4.0D. All sequence comparison parameters are set by theALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, .alpha.-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and abasic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S(“thioate”), P(S)S (“dithioate”), “(O)NR.sub.2 (“amidate”), P(O)R,P(O)OR′, CO or CH.sub.2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C.)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

The term “hepatocyte growth factor” or “HGF”, as used herein, refers toany native or variant (whether native or synthetic) HGF polypeptide thatis capable of inducing at least one, preferably all, steps in themulti-stage angiogenic process under conditions that permit such processto occur. Many agonists of HGF are known in the art, including HGFpolypeptide variants (such as deletion mutants comprising less than thecomplete form/amino acid sequence of native HGF protein, the delta5variant (see, for e.g., Miyazawa et al., Biochem. Biophys. Res. Comm.(1989), 163:967-973; Nakamura et al., Nature (1989), 342:440-443; Sekiet al., Biochem. and Biophys. Res. Commun. (1990), 172:321-327; Tashiroet al., Proc. Natl. Acad. Sci. U.S.A. (1990), 87:3200-3204; Okajima etal., Eur. J. Biochem. (1990), 193:375-381; U.S. Pat. No. 5,547,856)) andagonist antibodies (such as those described in U.S. Pat. No. 6,099,841).

Any term referring to a growth factor, angiogenic and other polypeptidesas used herein refers to any native or variant (whether native orsynthetic) of the polypeptide that is capable of effecting at least oneof the biological functions/activities of the native polypeptide, saidfunctions/activities being readily discernible in the context of thedescription of its use. Thus, for e.g., the term VEGF or bFGF wouldrefer to a native or variant form of the VEGF or bFGF protein,respectively, that retains the capability of inducing at least one,preferably all, steps in the multi-stage angiogenic process underconditions that permit such process to occur.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (for e.g., fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be human, humanized and/or affinitymatured.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody. In one embodiment, an antibody fragment comprisesan antigen binding site of the intact antibody and thus retains theability to bind antigen (preferably STC-1, which is preferably mammalianSTC-1, which is preferably human STC-1). In another embodiment, anantibody fragment, for example one that comprises at least a portion ofthe Fc region, retains at least one of the biological functions normallyassociated with the Fc region when present in an intact antibody, suchas FcRn binding, antibody half life modulation, ADCC function andcomplement binding.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Strict. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds (which ispreferably STC-1 or variant thereof as described herein). Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of STC-1, in particular withrespect to the modulatory activity of STC-1 in angiogenesis.

A “disorder” is any condition that would benefit from treatment with apolypeptide or method of the invention. This includes chronic and acutedisorders or diseases including those pathological conditions whichpredispose the mammal to the disorder in question. Non-limiting examplesof disorders to be treated herein include malignant and benign tumors;non-leukemias and lymphoid malignancies; neuronal, glial, astrocytal,hypothalamic and other glandular, macrophagal, epithelial, stromal andblastocoelic disorders; and inflammatory, immunologic and otherangiogenesis-related disorders.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some embodiments, polypeptides,polynucleotides, agonist and/or antagonist molecules (such asantibodies) of the invention are used to delay development of a diseaseor disorder.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result. A “therapeutically effective amount” of STC-1,agonist or antagonist may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theSTC-1, agonist or antagonist to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the STC-1, agonist or antagonist areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Vector Construction

Polynucleotide sequences encoding the polypeptides of the invention(including STC-1, antibodies) can be obtained using standard recombinanttechniques. Desired polynucleotide sequences may be isolated andsequenced from appropriate source cells, for example as illustrated inthe Examples below. Source cells for antibodies would include antibodyproducing cells such as hybridoma cells. Alternatively, polynucleotidescan be synthesized using nucleotide synthesizer or PCR techniques. Onceobtained, sequences encoding the immunoglobulins are inserted into arecombinant vector capable of replicating and expressing heterologouspolynucleotides in a host cell. Many vectors that are available andknown in the art can be used for the purpose of the present invention.Selection of an appropriate vector will depend mainly on the size of thenucleic acids to be inserted into the vector and the particular hostcell to be transformed with the vector. Each vector contains variouscomponents, depending on its function (amplification or expression ofheterologous polynucleotide, or both) and its compatibility with theparticular host cell in which it resides. The vector componentsgenerally include, but are not limited to: an origin of replication (inparticular when the vector is inserted into a prokaryotic cell), aselection marker gene, a promoter, a ribosome binding site (RBS), asignal sequence, the heterologous nucleic acid insert and atranscription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from a species compatible with the host cell are usedin connection with these hosts. The vector ordinarily carries areplication site, as well as marking sequences which are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322, a plasmid derived from an E.coli species. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM.TM.-11 may be utilized in making arecombinant vector which can be used to transform susceptible host cellssuch as E. coli LE392.

Either constitutive or inducible promoters can be used in the presentinvention, in accordance with the needs of a particular situation, whichcan be ascertained by one skilled in the art. A large number ofpromoters recognized by a variety of potential host cells are wellknown. The selected promoter can be operably linked to cistron DNAencoding a polypeptide of the invention (e.g., STC-1, antibodies) byremoving the promoter from the source DNA via restriction enzymedigestion and inserting the isolated promoter sequence into the vectorof choice. Both the native promoter sequence and many heterologouspromoters may be used to direct amplification and/or expression of thetarget genes. However, heterologous promoters are preferred, as theygenerally permit greater transcription and higher yields of expressedtarget gene as compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In some embodiments, each cistron within a recombinant vector comprisesa secretion signal sequence component that directs translocation of theexpressed polypeptides across a membrane. In general, the signalsequence may be a component of the vector, or it may be a part of thetarget polypeptide DNA that is inserted into the vector. The signalsequence selected for the purpose of this invention should be one thatis recognized and processed (i.e. cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processthe signal sequences native to the heterologous polypeptides, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group consisting of the alkaline phosphatase,penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB,PhoE, PelB, OmpA and MBP.

Prokaryotic host cells suitable for expressing polypeptides includeArchaebacteria and Eubacteria, such as Gram-negative or Gram-positiveorganisms. Examples of useful bacteria include Escherichia (e.g., E.coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species(e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans,Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus.Preferably, gram-negative cells are used. Preferably the host cellshould secrete minimal amounts of proteolytic enzymes, and additionalprotease inhibitors may desirably be incorporated in the cell culture.

Polypeptide Production

Host cells are transformed or transfected with the above-describedexpression vectors and cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ precipitation and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In preferred embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector, proteinexpression is induced under conditions suitable for the activation ofthe promoter. For example, if a PhoA promoter is used for controllingtranscription, the transformed host cells may be cultured in aphosphate-limiting medium for induction. A variety of other inducers maybe used, according to the vector construct employed, as is known in theart.

Polypeptides of the present invention expressed in a microorganism maybe secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therefrom. Cells maybe removed from the culture and the culture supernatant being filteredand concentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as fractionation on immunoaffinity orion-exchange columns; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; hydrophobic affinityresins, ligand affinity using a suitable antigen immobilized on a matrixand Western blot assay.

Polypeptide Purification

Polypeptides that are produced may be purified to obtain preparationsthat are substantially homogeneous for further assays and uses. Standardprotein purification methods known in the art can be employed. Thefollowing procedures are exemplary of suitable purification procedures:fractionation on immunoaffinity or ion-exchange columns, ethanolprecipitation, reverse phase HPLC, chromatography on silica or on acation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammoniumsulfate precipitation, and gel filtration using, for example, SephadexG-75.

Methods of the Invention

The invention provides various methods based on the finding that STC-1selectively modulates certain but not all aspects of angiogenesis. Forexample, the invention provides methods of diagnosis, such as tissuetyping, wherein STC-1 may be differentially expressed in one tissue ascompared to another, preferably in a diseased tissue as compared to anormal tissue of the same tissue type.

STC-1 polypeptides described herein may also be employed as therapeuticagents. These polypeptides can be formulated according to known methodsto prepare pharmaceutically useful compositions, whereby the polypeptideproduct hereof is combined in admixture with a pharmaceuticallyacceptable carrier vehicle. Therapeutic formulations are prepared forstorage by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid; low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

When in vivo administration of a polypeptide or agonist or antagonistthereof is employed, normal dosage amounts may vary from about 10 ng/kgto up to 100 mg/kg of mammal body weight or more per day, preferablyabout 1 μg/kg/day to 10 mg/kg/day, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. Nos.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

Where sustained-release administration of a polypeptide is desired in aformulation with release characteristics suitable for the treatment ofany disease or disorder requiring administration of the polypeptide,microencapsulation of the polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhIFN—), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology. 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

The sustained-release formulations of these proteins were developedusing polylactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 141.

This invention encompasses methods of screening compounds to identifythose that mimic STC-1 (agonists) or prevent the effect of STC-1(antagonists). Screening assays for antagonist drug candidates aredesigned to identify compounds that bind or complex with the STC-1polypeptides, or otherwise interfere with the interaction of STC-1 withother cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with an STC-1 polypeptide under conditions and for atime sufficient to allow these two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, an STC-1 polypeptide or the drug candidate is immobilized ona solid phase, e.g., on a microtiter plate, by covalent or non-covalentattachments. Non-covalent attachment generally is accomplished bycoating the solid surface with a solution of the immobilized component(for e.g., STC-1 polypeptide) and drying. Alternatively, an immobilizedantibody, e.g., a monoclonal antibody, specific for the component to beimmobilized (for e.g., STC-1 polypeptide) can be used to anchor it to asolid surface. The assay is performed by adding the non-immobilizedcomponent, which may be labeled by a detectable label, to theimmobilized component, e.g., the coated surface containing the anchoredcomponent. When the reaction is complete, the non-reacted components areremoved, e.g., by washing, and complexes anchored on the solid surfaceare detected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to a STC-1polypeptide, its interaction with the polypeptide can be assayed bymethods well known for detecting protein-protein interactions. Suchassays include traditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a STC-1 polypeptide andother intra- or extracellular components can be tested as follows:usually a reaction mixture is prepared containing the product of thegene and the intra- or extracellular component under conditions and fora time allowing for the interaction and binding of the two products. Totest the ability of a candidate compound to inhibit binding, thereaction is run in the absence and in the presence of the test compound.In addition, a placebo may be added to a third reaction mixture, toserve as positive control. The binding (complex formation) between thetest compound and the intra- or extracellular component present in themixture is monitored as described hereinabove. The formation of acomplex in the control reaction(s) but not in the reaction mixturecontaining the test compound indicates that the test compound interfereswith the interaction of the test compound and its reaction partner.

To assay for antagonists, the STC-1 polypeptide may be added to a cellalong with the compound to be screened for a particular activity and theability of the compound to inhibit the activity of interest in thepresence of the STC-1 polypeptide indicates that the compound is anantagonist to the STC-1 polypeptide. To assay for agonists, a candidateagonist compound can be contacted with a cell known to respond to theSTC-1 polypeptide, and assessed for an activity associated with theSTC-1 polypeptide. The assay could also be performed in the presence ofthe STC-1 polypeptide itself to assess ability of the candidate compoundto enhance the activity of STC-1.

More specific examples of potential agonists and antagonists includeantibodies including, without limitation, poly- and monoclonalantibodies and antibody fragments, single-chain antibodies,anti-idiotypic antibodies, and chimeric or humanized versions of suchantibodies or fragments, as well as human antibodies and antibodyfragments. Alternatively, a potential antagonist may be a closelyrelated protein, for example, a mutated form of the STC-1 polypeptidethat recognizes an STC-1 binding partner but imparts no effect, therebycompetitively inhibiting the action of the STC-1 polypeptide.

Another potential STC-1 polypeptide antagonist is an antisense RNA orDNA construct prepared using antisense technology, where, e.g., anantisense RNA or DNA molecule acts to block directly the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes theSTC-1 polypeptide, is used to design an antisense RNA oligonucleotide offrom about 10 to 40 base pairs in length. A DNA oligonucleotide isdesigned to be complementary to a region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res., 6:3073(1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., Science,251:1360 (1991)), thereby preventing transcription and the production ofthe STC-1 polypeptide. The antisense RNA oligonucleotide hybridizes tothe mRNA in vivo and blocks translation of the mRNA molecule into theSTC-1 polypeptide (antisense—Okano, Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRCPress: Boca Raton, Fla., 1988). The oligonucleotides described above canalso be delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of the STC-1 polypeptide. Whenantisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

Potential agonists and antagonists include small molecules that bind tothe active site, the binding site of STC-1 on another molecule, or otherrelevant binding site of the STC-1 polypeptide, thereby mimicking,enhancing or blocking the normal biological activity of the STC-1polypeptide. Examples of small molecules include, but are not limitedto, small peptides or peptide-like molecules, preferably solublepeptides, and synthetic non-peptidyl organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

Anti-STC-1 Antibodies

The present invention provides methods comprising use of anti-STC-1antibodies. Exemplary antibodies include polyclonal, monoclonal,humanized, bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-STC-1 antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the STC-1 polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-STC-1 antibodies may, alternatively and preferably, bemonoclonal antibodies. Monoclonal antibodies may be prepared usinghybridoma methods, such as those described by Kohler and Milstein,Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, orother appropriate host animal, is typically immunized with an immunizingagent to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include the STC-1 polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed againstSTC-1. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

Antibodies can also be generated by screening phage display librariesfor antibodies or antibody fragments that bind with suitable/desiredaffinity to a target antigen such as STC-1 or an STC-1 variant. Suchtechniques are well known in the art, for e.g., as disclosed in U.S.Pat. Nos. 5,750,373; 5,780,279; 5,821,047; 6,040,136; 5,427,908;5,580,717, and references therein.

3. Human and Humanized Antibodies

The anti-STC-1 antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

The antibodies may also be affinity matured using known selection and/ormutagenesis methods as described above. Preferred affinity maturedantibodies have an affinity which is five times, more preferably 10times, even more preferably 20 or 30 times greater than the startingantibody (generally murine, humanized or human) from which the maturedantibody is prepared.

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities can befor the STC-1 polypeptide, the other one can be for any other antigen,and preferably for a cell-surface protein or receptor or receptorsubunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.175:217-225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on aSTC-1 polypeptide herein. Alternatively, an anti-STC-1 polypeptide armmay be combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing STC-1. Bispecific antibodies may also be used tolocalize cytotoxic agents to cells which express the STC-1 polypeptide.These antibodies possess a STC-1 binding arm and an arm which binds acytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA,or TETA. Another bispecific antibody of interest binds the STC-1polypeptide and further binds tissue factor (TF).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

9. Compositions of Antibodies

Antibodies specifically binding STC-1 polypeptide, as well as othermolecules identified by the screening assays disclosed hereinbefore, canbe administered for the treatment of various disorders in the form ofcompositions, wherein in some embodiments the compositions also comprisea carrier (for e.g., a pharmaceutically acceptable carrier).

If the STC-1 polypeptide is to be targeted intracellularly and wholeantibodies are used as inhibitors, internalizing antibodies arepreferred. However, lipofections or liposomes can also be used todeliver the antibody, or an antibody fragment, into cells. Whereantibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). Theformulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

EXAMPLES Example 1 Materials and Methods

Reagents: Human umbilical vein endothelial cells (HUVECs) were purchasedfrom Clonetics (San Diego, Calif.) and maintained in Endothelial GrowthMedium (EGM) (Clonetics) supplemented to a final concentration of 10%fetal bovine serum. Other reagents included type 1 rat tail collagen(Upstate Biotechnology. Lake Success, N.Y.), recombinant bFGF(Collaborative Biomedical Products, Bedford, Mass.), recombinant VEGFand HGF (Genentech, South San Francisco, Calif.). All other cell culturereagents were from Gibco-BRL (Gaithersburg, Md.).Isolation of STC-1 and Construction of Expression Vectors: cDNA cloneswere isolated from a human endothelial cDNA library and sequenced intheir entirety. Fc fusion proteins (immunoadhesins) were prepared byfusion of the entire open reading frame of STC-1 in frame with the Fcregion of human IgG1 using the baculovirus vector pHIF, a derivative ofpVL1393 purchased from Pharmingen. Fusion proteins were transientlyexpressed in Sf9 insect cells and purified over a protein A column.STC-1 was also expressed as a C-terminal His tag fusion in Escherichiacoli and the denatured protein used for immunization. The identities ofthe purified proteins were verified by N-terminal sequence analysis.Preparation of monoclonal antibodies to STC-1: Ten Balb/c mice (CharlesRiver Laboratories, Wilmington, Del.) were hyperimmunized withrecombinant polyhistidine-tagged (HIS8) human STC-1 expressed in E. coli(Genentech, Inc. South San Francisco, Calif.) in Ribi adjuvant (RibiImmunochem Research, Inc., Hamilton, Mo.). B-cells from five micedemonstrating high anti-STC-1 antibody titers were fused with mousemyeloma cells (X63.Ag8.653; American Type Culture Collection, Rockville,Md.) using a modified protocol analogous to one previously described(3,4). After 10-14 days, the supernatants were harvested and screenedfor antibody production by direct enzyme-linked immunosorbent assay(ELISA). Five positive clones, showing the highest immunobinding afterthe second round of subcloning by limiting dilution, were injected intoPristaneprimed mice (5) for in vivo production of MAb. The ascitesfluids were pooled and purified by Protein A affinity chromatography(Pharmacia fast protein liquid chromatography [FPLC]; Pharmacia,Uppsala, Sweden) as previously described (3). The purified antibodypreparations were sterile filtered (0.2-μm pore size; Nalgene, RochesterN.Y.) and stored at 4° C. in phosphate buffered saline (PBS).

Antibody clones were deposited with the ATCC under the Budapest Treaty.American Type Culture Collection (ATCC) is located at 10801 UniversityBoulevard, Manassas, Va. 20110-2209. Details of the deposits are asfollows:

Clone designation Deposit Date ATCC Deposit No. 3E10.1F5.1D11 Mar. 28,2003 PTA-5100 21F4.2F11.1H12 Mar. 28, 2003 PTA-5103 1F10.2F12.1E7 Mar.28, 2003 PTA-5101 18F1.1E6.1B10 Mar. 28, 2003 PTA-5099 12C3.2F3.2D8 Mar.28, 2003 PTA-5102ELISA for STC-1: High-binding, flat-bottom polypropylene 96-well plates(NUNC, Naperville, Ill.) were coated overnight at 4° C. with 100 μLmonoclonal STC-1 antibody 2734 (clone 21F4.2F11.1H12, ATCC Deposit No.PTA-5103) (250 ng/mL). The plates were washed (PBS containing 0.05%Tween), blocked (PBS containing 0.5% BSA), and washed again beforeadding 100 μL of supernatant or STC-1 onto duplicate wells. Aftersubsequent washing steps, a second biotinylated STC-1 monoclonalantibody (2733 (clone 12C3.2F3.2D8, ATCC Deposit No. PTA-5102); 250ng/mL) was added to the wells. After 2 hr incubation and a wash step, a1:10,000 dilution of Streptavidin-HRP (Amersham, Gaithersburg, Md.) wasadded to the plates. Tetramethyl benzidine (TMB, Kirkegaard & Perry,Gaithersburg, Md.) was added followed by 1 M phosphoric acid and theabsorbance at 450 nm determined (Spectra Max 250 Molecular Devices,Sunnyvale, Calif.). The minimum level of STC-1 that could be reliablydetected by the ELISA was 20 pg/mL.RNA Isolation and Quantitative Reverse Transcriatase-Polymerase ChainReaction (Tagman): Tri-Reagent-LS (Molecular Research Center,Cincinnati, Ohio) was added to the cells and total RNA was extractedaccording to manufacturer's protocols. Gene-specific oligonucleotideprimer pairs and a specific probe (labeled with a fluorescent dye at the5′ end and a quencher fluorescent dye at the 3′ end) were designed usingOligo 4.0 software (National Biosciences, Plymouth, Minn.) and levels ofSTC-1 mRNA determined by real time quantitative PCR (Tagman) aspreviously described (6). Sequences for primers and probe were asdescribed in Table 1 (under Gene name “Stanniocalcin precursor”) of Kahnet al., Am. J. Pathology (2000), 156(6): 1887-1900.Western Blotting: Equal amounts of supernatants were denatured andloaded onto 10% Tris-Glycine gels. Following electrophoresis, theproteins were transferred to nitrocellulose membranes using a PantherHEP-3 semi-dry blotter (Owl Scientific Separations-Daigger, Vernon HillsIll.). STC-1 was detected using STC-1 antibody 2734 (clone21F4.2F11.1H12, ATCC Deposit No. PTA-5103) and the ECL detection method(Amersham Pharmacia, Piscataway).Culture of Cells: HUVECs were routinely grown on gelatin (1 μg/mL)coated plates in EGM media. Drugs and growth factors were added to themedia and prewarmed to 37° C. before addition to the HUVECs. Collagengels containing HUVECs were prepared as described previously (1). Thegels were overlaid with 1× basal media (Medium 199 supplemented with 1%fetal bovine serum, 1% ITS (Insulin, selenium and transferring, source)2 mM L-glutamine) and 100 U/mL penicillin, and 100 U/mL streptomycin)containing 200 ng/mL HGF and 200 ng/mL VEGF to elicit tube formation aspreviously described (1). For “film” experiments endothelial cells wereseeded on the surface of a collagen gel and incubated in the identicalmedia as that described for the gel experiments. To evaluate endothelialmorphogenesis on Matrigel (Collaborative Research, Bedford, Mass.),cells were incubated in 1× basal media in the presence of variousfactors as described. Network formation was quantitated at 8 hours byphotographing 3 random fields of each well, then determining the totalnetwork area per field using Openlab 2.5 software (Improvision, Bedford,Mass.).Cell Migration Assay: HTS multiwell insert 24 well plates (BDBiosciences, Bedford, Mass.) were coated with cell attachment factor (BDBiosciences) on the bottom layer and type 1 collagen on the membranesurface. 25,000 cells were seeded into each chamber and incubated for 18h at 37° C./5% CO₂. The collagen and unmigrated cells were scraped offthe membrane surface with a plastic pasteur pipette and then all mediawas aspirated. Absolute methanol was added to the membrane and membranesfixed at room temperature for 30 minutes. The methanol was aspirated offand a 10 μM solution of YO-PRO-1 (Molecular Probes, Eugene, Oreg.) wasadded. Cells were counted under FITC optics using OpenLab 2.5.Proliferation Assay: 5,000 cells were seeded onto gelatin coated 96 wellplates and incubated overnight with EGM. The cells were then starved for3 days with M199 containing 1% FBS, 2 mmol/L L-glutamine, 100 U/mLpenicillin, and 100 U/mL streptomycin. 20 ng/mL of various growthfactors were added to the starvation media and the cells were incubatedfor 4 days. Alamar blue solution (Biosource International, Camarillo,Calif.) was added to the wells in an amount equal to 10% of the culturevolume and incubated for 4-6 hours at 37° C./5% CO₂. The plates wereread on a Spectra Max Gemini (Molecular Devices, Sunnyvale, Calif.) withthe OD excitation at 535 nm and emission at 590 nm.Femoral Ligation Surgery: Femoral artery ligation was performed underIsoflurane (Aerrane, Fort Dodge) inhalation anesthesia on Male C57/BI6Jmice (Charles River) (8-10 weeks). Briefly, the femoral artery wasisolated at the level of the inguinal ligament and ligated with 7-0 silksuture (Ethicon, Somerville, N.J.). Animals were allowed to recover on awarm water heating pad until ambulatory. Total RNA was isolated from thegastrocnemius muscle of both the ligated and sham animals. Six animalswere used for the control (sham) and six animals for the ligated grouprespectively for each time point.Effects of STC-1 on HGF induced c-met phosphorylation: Confluent HYVEcwere incubated overnight in Basal Medium. HUVEc monolayers werepretreated with native or boiled STC-1 (5 μg/ml) for 30 min in DMEsupplemented with 0.5% bovine serum albumin. HGF was then added (10ng/ml) and cells incubated for 15 minute incubation at 37° C. Afteraddition of lysate buffer (PBS supplemented with 1% triton, proteaseinhibitor mix (Sigma) and phosphatase inhibitor mix (Sigma), lysateswere immunoprecipitated with an antibody to c-met (C-28, Santa CruiseBiotechnology). Precipitates were resuspended in lysis buffer and loadedonto 10% Tris-Glycine gel, and western blots prepared as above.Phosphorylated c-met was detected with 4G10 (Upstate Biotechnology,N.Y.).Data analysis: Numerical data are expressed as the mean±SEM and the nfor each experiment is provided in the figure legends or text. Todetermine statistical significance data were first evaluated by ANOVA,followed by Student's t-test for non-paired values. A p-value of <0.05was accepted as significant.

RESULTS

Regulation of STC-1 Production in Endothelial Cells in MonolayerCulture.

To evaluate the effects of various cytokines and growth factors on therelease of STC-1 from HUVEC, we developed a ELISA based assay (seemethods for details) which was capable of measuring STC-1 levels as lowas 20 pg/ml. There was no detectable STC-1 release from unstimulatedHUVEC (not shown). To survey for possible effects of various cytokinesand growth factors on STC-1 release, confluent endothelial cellscultured in 96-well tissue culture plates were incubated with thesefactors for 24, 48 and 72 hours, and STC-1 levels determined in the cellsupernatants. The majority of factors evaluated had either no effect(e.g. VEGF, TGFβ, bradykinin, histamine, and TNFα) or very modesteffects (IL-1β) on STC-1 release at these time points. Theconcentrations shown in Table 1 are the highest concentration tested(for each drug we tested the indicated dose and at least two lower doses(e.g. 1:10 and 1:100) of that shown). Of the growth factors examined,only bFGF and HGF stimulated significant STC1 release. The cytokinesIL-6 and IL-4 also stimulated STC-1 release.

TABLE 1 Effects of various agonists on the release of STC-1 by HUVEC.HUVEC monolayers were incubated with the indicated agonists and aliquotsremoved at 24, 48 and 72 hr and analyzed for STC-1 by ELISA. Data shownare the mean values (n = 4). STC-1 (ng/ml) Stimuli 24 hr 48 hr 72 hrMedia <.02 <.02 <.02 Calcium <.02 <.02 ND Ionophore 10 mg/ml Bradykinin(1 μM) <.02 <.02 ND Histamine (1 μM) <.02 <.02 ND VEGF (400 ng/ml <.02<.02 <.02 TGF β (50 ng/ml) <.02 <.02 <.02 IFNγ (25 ng/ml) <.02 <.02 <.02bFGF (400 ng/ml <.02 <.02 .092 HGF (400 ng/ml <.02 <.02 .112 TNFα (10ng/ml) <.02 <.02 .06 IL-6 (25 ng/ml <.02 .023 .114 IL-1 (10 ng/ml <.02.027 .027 IL-4 25 ng/ml <.02 .025 .122 PMA (100 ng/ml) .023 .057 .183Levels of STC-1 mRNA and protein secretion are much greater in 3-Dcultures compared to 2-D cultures. Equal numbers (1.5×10⁷) of HUVEc wereplated either onto type I collagen gels (“film”) or suspended in type Icollagen gels (gel) and incubated with HGF and VEGF (200 ng/ml) forvarious times as indicated in FIG. 1. It should be noted that thecombination of HGF and VEGF was required in 3D gels for survival—asdescribed previously, neither growth factor alone was capable ofsupporting survival and tubulogenesis in 3D collagen gels (1). The mRNAlevels (FIG. 1A) for STC-1 rose dramatically in the gel versus filmenvironment, such that at 24 hr, the mRNA levels for STC-1 were 1020fold higher in the 3-D cultures. Levels of STC-1 protein (FIG. 1B) inthe supernatants were 2 to 6 fold higher in the 3-D compared to filmcultures. Since the cells are embedded in 3D collagen gels, any proteinin the supernatants must be “released” from the gel in order to bedetected, and may account for the discrepancy in relative ratios of mRNAversus protein.Effects of STC-1 on endothelial proliferation. STC-1 (0.001-1 μg/ml) hadno effect on bFGF (10 ng/ml), VEGF (10 ng/ml) or HGF (10 ng/ml)stimulated endothelial proliferation (data not shown). Additionally,STC-1 did not stimulate endothelial proliferation when tested in theabsence of growth factors (not shown).Effects of STC-1 on HGF induced endothelial migration: HGF is a knownpotent stimulus for endothelial migration. We, therefore, determined therole of STC-1 in HGF induced endothelial cell migration. As shown inFIG. 2A, rSTC1 (Ig fusion protein) when added to the Boyden chambers,markedly inhibited the migratory response of the endothelial cells toHGF. Denaturation of the recombinant protein by boiling completelyeliminated this inhibitory activity. Additionally, the inhibitoryeffects of STC-1 were not observed at lower (2.5 ng/mL) concentrationsof the protein. The inhibitory effects of STC-1 on HGF cell migrationwere also blocked by inclusion of 25 μg/mL of the anti-STC-1monoloclonal antibody 2734 (clone 21F4.2F11.1H12, ATCC Deposit No.PTA-5103) (FIG. 2B) in contrast to the lack of effect of an isotypematched non-imnune IgG. To further evaluate the selective effects ofSTC-1 on endothelial cell migration, we evaluated the effects of rSTC-1on bFGF (10 ng/mL) and VEGF (10 ng/mL) induced endothelial cellmigration. These doses of bFGF and VEGF elicited a similar magnitude ofcell migration as 20 ng/mL HGF, yet none of the STC-1 reagents tested(native STC-1, boiled STC-1, STC1 monoclonal antibody 2734 (clone21F4.2F11.1H12, ATCC Deposit No. PTA-5103) (25 μg/mL)) had a significanteffect on the migratory response to bFGF or VEGF (data not shown).Effects of STC-1 on HGF induced endothelial morphogenesis To evaluatethe possible effects of STC-1 on endothelial morphogenesis, we testedthe effects of native rSTC-1 and boiled STC-1 on HGF induced endothelialbranching network formation on growth factor depleted Matrigel. In thismodel, incubation of endothelial cells without a growth factor such asHGF results in little or no cord formation (not shown). Addition of HGF(20 ng/ml) results in an elaborate network of endothelial branchingstructures (FIG. 3A). Addition of rSTC-1 (1 μg/ml) markedly reduced theoverall area of endothelial networks (FIG. 313) and the structures thatdid form were discontinuous and poorly formed. Heat denaturationabrogated the effects of rSTC-1 (FIG. 3C).Effects of STC-1 on HGF induced c-met phosphorylation. HGF induced thephosphorylation of c-met (FIG. 4). Pretreatment or cotreatment of HUVECwith 5 μg/ml STC-1 did not reduce the phosphorylation response of c-metto added HGF (FIG. 4), suggesting that STC-1 did not inhibit HGF bindingto its receptor. To determine possible effects downstream of c-metphosphorylation, the effects of STC-1 on HGF-induced FAK activation wasalso examined. The data indicated that maximal FAK phosphorylation byHGF in HUVEC occurred at 60 min (data not shown). Pretreatment of theendothelial cells with STC-1 abrogated HGF-induced phosphorylation ofFAK (data not shown). Although difficult to detect, the modest FAKphosphorylation induced by bFGF or VEGF (data not shown) was notinhibited by pretreatment with STC-1.Expression of STC-1 in the hind-limb ischemia model of angiogenesis. Wealso evaluated the expression of STC-1 in the mouse femoral ligationassay. In this model, the femoral artery is ligated, dramaticallyreducing blood flow to the lower limb including the gastrocnemius muscle(7). This hypoxic insult results in the rapid development of new vesselsin the hypoxic muscle as a component of the spontaneous recovery ofperfusion which is also associated with proximal arteriogenesis (8,9).Because the newly developing vessels are not readily visualized in thisassay, we also measured the mRNA levels of the endothelial marker, CD31as an index of new vessel formation. As shown in FIG. 5A, both CD31 mRNAand STC-1 mRNA expression peaked at day 3 and then returned to lowerlevels at later time points. We also measured the mRNA levels of HGF.Interestingly, the expression of HGF also increased markedly in this invivo model, peaking at day 3 (FIG. 5B)

Discussion

Stanniocalcin 1, a secreted protein, was first identified in fish, whereit has been shown to regulate calcium and phosphate homeostasis (10,11).A unique feature of stanniocalcin 1 is its lack of homology to any otherproteins, other than stanniocalcin 2 to which it is 34% identical at theamino acid level. The human homolog of stanniocalcin 1 is 73% homologousto the salmon protein. However, in contrast to the fish, wherestanniocalcin 1 is exclusively localized to the organ of Stannius, themammalian homolog exhibits a much broader expression profile. Thefunction of the mammalian stanniocalcin 1 is generally poorlyunderstood, although roles in calcium and phosphate homeostasis (12-14)and ovarian function (15) have been suggested. The majority of“functions” that have been attributed to mammalian stanniocalcin 1 areassociative, i.e. they have been based on expression data or cellularlocalization. The data described herein provide the first “biologicalactivity” of stanniocalcin 1 on mammalian cells. The activities in theMatrigel and migration assay, which are well accepted in the art asmodels for the multi-staged angiogeneis process, provide unexpected andimportant evidence for a role for stanniocalcin vascularization andangiogeneis-associated signaling and function.

We first identified stanniocalcin 1 as one of the genes thatdemonstrated marked upregulation in endothelial cells undergoingtubulogenesis (6), suggesting a possible role in angiogenesis. In situstudies demonstrated that the expression of stanniocalcin 1 was highlyfocal—high levels of expression were observed in small to large vesselsat the periphery of lung and colon carcinomas and inflamed appendix(2,6). Moreover, STC-1 mRNA was markedly increased in rat corneasimplanted with hydron pellets containing VEGF compared to vehiclecontrols (2). Examination of the phenotype of stanniocalcin 1 transgenic(Tg) mice (using a muscle specific promoter) revealed that thestanniocalcin 1 Tg mice were smaller than their wild type littermates,yet baseline organ vascularity as well as induction of increasedvascular density following femoral ligation were enhanced (14). Weherein report the surprising observation that stanniocalcin-1 is anautocrine modulator of HGF-induced endothelial migration andmorphogenesis (cord formation). These effects were selective toparticular angiogenic factors, in particular HGF, because the responsesof endothelial cells to either VEGF or bFGF were not modulated in thesein these assays. Of the growth factors and cytokines examined, HGF wasthe most potent inducer of stanniocalcin 1 secretion. In vivo dataprovide further support for this conclusion. In an in vivo model ofphysiological angiogenesis, the mouse femoral ligation model, theexpression profile of stanniocalcin 1 mRNA was similar to that of theendothelial marker CD31, and moreover, the peak expression ofstanniocalcin 1 mRNA was preceded by peak expression of HGF.

The mechanism of the selective inhibition of the angiogenic action of afirst angiogenic factor but not of a second angiogenic factor onendothelial cells is intriguing—stanniocalcin did not inhibit HGFinduced endothelial proliferation or c-met phosphorylation, ruling out adirect inhibitory effect on HGF binding to its receptor. However, theselective inhibitory effects of STC-1 on HGF mediated responses (versusbFGF or VEGF) strongly suggest that STC-1 modulates specific features ofHGF signaling, and thus its angiogenic effector pathway. STC-1 reducedHGF-induced FAK phosphorylation, consistent with the notion that STC-1interferes with one or more downstream signaling pathways activated bythe c-met receptor. Moreover, the effects of STC-1 appears to beselective for HGF since bFGF and VEGF-induced FAK phosphorylation is notinhibited. FAK is a focal adhesion kinase that is localized in focaladhesions and has been shown to play an important role inintegrin-mediated cellular functions; FAK activation is also linked toHGF-induced cell motility. The data described herein are consistent witha modulator role of STC-1 in angiogenesis, possibly serving as a “Stopsignal” or stabilization factor contributing to the maturation of newlyformed blood vessels. Although HGF is not a selective endothelialmitogen or motogen, it is a very potent angiogenic/growth agent (16,17)and the expression of both HGF and its receptor c-met are known to beupregulated in both physiological (18,19) and pathological angiogenesis(20,21). Therefore, as described herein, STC-1 provides a unique andpotentially highly advantageous target for selective therapeuticmodulation of angiogenesis. In one aspect, it is an advantageous targetfor selective modulation of certain, but not all, steps in themulti-stage angiogenic process.

STC-1 receptors, at least in the liver and kidney, have been reported tobe present both on the plasma membrane and in the mitochondria (22).Furthermore, despite being a secreted protein, STC-1 is sequestered inthe mitochondria (22) and has been proposed to play a role in theregulation of cellular metabolism (14,22). Thus the elevated expressionof STC-1 during angiogenesis may play additional roles in the metabolicrequirements of endothelial cells and other cells involved in theformation of new blood vessels.

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1. A method of selectively inhibiting an angiogenic process promoted bya first angiogenic factor, but not the angiogenesis process promoted bya second angiogenic factor, comprising administering to a cell exposedto both factors an effective amount of STC-1 (SEQ ID NO: 2), wherein theSTC-1 inhibits at least one step of the angiogenic process promoted bythe first angiogenic factor but not the second angiogenic factors,wherein the first angiogenic factor is hepatocyte growth factor and thesecond angiogenic factor is selected from the group consisting of:fibroblast growth factor and vascular endothelial growth factor, andwherein the angiogenic process is cell migration.
 2. The method of claim1, wherein the first and second angiogenic factors both are capable ofinducing endogenous expression of STC-1.
 3. The method of claim 1,wherein STC-1 does not inhibit cell proliferation.